MUTATION  IN  THE  GENUS  ALTERNARIA 


BY 

ORDA  ALLEN  PLUNKETT 

A.  B.  Wabash  College,  1920 


THESIS 

SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS 
FOR  THE  DEGREE  OF  MASTER  OF  ARTS  IN  BOTANY 
IN  THE  GRADUATE  SCHOOL  OF  THE 
UNIVERSITY  OF  ILLINOIS, 

1922 


URBANA,  ILLINOIS 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/mutationingenusaOOplun 


I z)  (^  fa 

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UNIVERSITY  OF  ILLINOIS 
THE  GRADUATE  SCHOOL 


Jlay  -igi  2 


I HEREBY  RECOMMEND  THAT  THE  THESIS  PREPARED  UNDER  MY 
SUPERVISION  BY Orda  Allen  Plunkett 


ENTITLED. 


Mutation  in  the  Genua  Alt exnaria^ 


BE  ACCEPTED  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR 
THE  DEGREE  OF 


Recommendation  concurred  in* 


Committee 


on 


Final  Examination* 


'‘Required  for  doctor’s  degree  but  not  for  master’s 


. 'v  yi  O 

VOVjo 


■ 4. 


' 


Table  of  Contents 

Page 

I.  Introduction  1 

II.  The  present  status  of  the  mutation  question  in 

fungi  and  bacteria  3 

III.  Source  of  materials  20 

IV.  Methods  employed  for  isolation  and  growth  21 

Appearance  of  mutations  22 

Modifications  24 

V.  Methods  employed  for  contrast  studies  of  mutants 

and  originals  35 

Color  of  colonies  on  agar  36 

Zonation  of  colonies  27 

Rate  of  growth  20 

Color  of  mycelium 20 

Color  of  medium 29 

Aerial  mycelium  29 

Length  of  conidia 30 

Width  of  conidia  32 

Cross  septa  33 

Longitudinal  septa  35 

Color  of  conidia  36 

Conidial  production  37 

Permanency  of  mutants  38 

Frequency  of  mutation  38 

VI.  Conclusions  39 

VII.  Literature  cited  41 

VIII.  Explanation  of  plates  45 


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Mutation  in  the  Genus  Alternaria 
I.  Introduction 

The  present  study  arose  from  an  interest  which  the 
writer  had  in  the  appearance  of  a sector  of  decidedly  different 
color  in  a supposedly  pure  culture  of  Alternaria  growing  in  a 
Petri  dish  on  corn-meal  agar.  Single  spores  of  this  culture 
were  grown  and  the  colonies  examined  for  further  evidence  of 
this  phenomenon.  It  was  found  that  quite  often  in  these  single- 
spore cultures  variant  sectors  were  formed,  which  retained  the 
characteristic  variation  and  remained  different  from  the  parent 
colony  throughout  many  transfers.  This  led  to  a tentative  assump- 
tion that  these  variant  sectors  represented  mutants  or  saltants 
of  more  or  lees  permanent  nature,  and  a more  detailed  study  of 
this  phenomenon  was  therefore  undertaken. 

The  writer  has  cultured  on  corn-meal  agar,  some  15 
strains  of  Alternaria  isolated  from  different  hosts,  to  see  if 
the  phenomenon  of  variant  sectors  is  equally  common  in  all  these 
species  or  races.  These  strains  have  all  been  grown  from  single 
spores  to  avoid  the  possibility  of  error  arising  from  mixed  cul- 
tures. 

The  variants  found  in  the  study  of  several  strains  have 
been  carefully  examined  and  compared  in  relation  to  each  other 


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and  to  the  original  form. 

II.  The  Present  Status  of  the  Mutation  Question 
in  Fungi  and  Bacteria 

Bud  variations,  vegetative  variations,  sports,  mutations 
and  saltations  have  been  known  and  studied  among  the  higher  plants 
for  eome  time.  In  these  cases  such  variations  may  or  may  not  be 
connected  with  an  intervening  sexual  act.  That  similar  cases 
appear  among  bacteria  and  fungi  is  evident  from  a number  of  arti- 
cles bearing  on  this  subject  published  in  the  last  few  years. 

The  mutations  occurring  in  bacteria  have  been  divided 
by  Dobell  (16)  into  two  classes,  ’’those  in  which  the  change  is 
functional  (changes  in  the  power  of  producing  ferments  or  pigment e) 
and  those  in  which  the  change  is  structural”.  The  greater  number 
of  mutations  recorded  for  bacteria  belong  to  the  first  class. 

Massini  (26)  obtained  from  a case  of  enteritis  an  organ- 
ism which  at  first  produced  whitish  colonies  on  Endo's  medium. 
After  a time,  however,  red,  daughter  colonies  appeared  within  the 
parent  colony.  Massini  convinced  himself,  by  carefully  plating 
out  the  original  culture,  and  by  other  means,  that  he  had  not 
been  working  with  a mixed  culture.  The  organism  in  the  red  daugh- 
ter colonies  had  permanently  acquired  the  power  to  ferment  lactose 
and  always  produced  red,  and  never  white  colonies.  Even  after 
transplanting  to  other  media,  free  from  lactose,  they  never  lost 
this  oower.  The  trohosus-l ike  original  therefore,  had  given  rise 


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to  a number  of  new  individuals  which  closely  resembled  the  original, 
but  which  had  acquired  the  power  to  ferment  lactose.  The  white 
parts  of  the  colonies  containing  the  red,  daughter  colonies  be- 
haved exactly  like  the  original  when  transplanted.  The  white  race 
was  therefore  constantly  undergoing  a partial,  mutation  into  a pure 
red  race. 

Twort  (42)  recorded  independently  that  certain  coli- 
typhosus  organisms  were  able  to  acquire  the  power  to  ferment  cer- 
tain sugars  if  grown  in  them  for  a sufficiently  long  time.  Ey 
this  method  he  was  able  to  get  a strain  of  B.  typhosus  which  could 
ferment  lactose  and  dulcite.  The  organism  which  had  acquired  this 
power  retained  it  permanently,  even  after  passage  through  a guinea- 
pig  and  cultivation  in  a dulcite-free  medium. 

Burke  (10),  Sauerbeck  (33),  Benecke  (4)  and  Kowalenko 
(23)  have  isolated  organisms  similar  to  the  one  studied  by  Maesini 
(26).  Their  results  check  his  and  in  addition  make  an  important 
addition  to  them,  since  they  succeeded  in  testing  individual  organ- 
isms. 

Kowalenko  (23)  found  that  a single  individual , which  at 
the  outset  was  unable  to  split  lactose,  produced  in  the  presence 
of  this  substance  offspring,  which  were  in  part  like  itself,  and 
in  part  able  to  eplit  lactose.  The  only  objection  to  Massini’s 
results  was  therefore  removed.  Single  individuals  from  white 
colonies  gave  rise  to  mutating  races;  single  individuals  from  "red" 
colonies  retained  their  characters  in  following  generations. 


if 


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Their  properties  were  unaltered  by  passage  through  animals,  by 
changes  of  temperature,  or  by  phenol  or  other  drugs. 

Muller  (27)  demonstrated  that  all  typical  races  of 
B.  typhosus  are  unable  to  ferment  rhamnose.  When  grown  in  a med- 
ium containing  this  sugar,  however,  the  colonies  develop  daughter 
colonies  (nodules)  consisting  of  individuals  which  have  permanent- 
ly acquired  the  power  of  splitting  rhamncse.  A single  non-rhamnose 
splitting  individual  gives  rise  in  the  presence  of  this  substance 
to  offspring  which  are  partly  like  itself  and  partly  able  to  fer- 
ment rhamnose.  Mutations  of  this  sort  invariably  occur  from  single 
individual  cultures  and  under  no  conditions  do  the  rhamnose  split- 
ting individuals  lose  their  power. 

SchrOter  and  Gtltjahr  (35)  record  the  following  observa- 
tions. A race  Y of  coli  typhosus-group  organisms  cannot  ferment 
maltose.  After  cultivation  in  a medium  containing  this  sugar, 

4 . 

however,  it  partially  acquires  the  power,  thus  coming  to  resemble 
Flexner’s  bacilli.  They  found  similar  changes  in  related  organ- 
isms; for  example,  Shiga-Kruse  bacilli  may  acquire  the  power  of 
splitting  both  maltose  and  saccharose  when  grown  in  media  contain- 
ing these  sugars.  The  change  appears  to  be  permanent,  for  once 
acquired  by  the  race  the  property  is  never  lost. 

Sobernheim  and  Seligmann  (36)  have  isolated  an  oa^nism 
which  behaves  exactly  like  Mass  ini’s  B.  coli  mutabile.  In  addi- 
tion they  record  that  they  have  obtained  four  different  pure  races 
from  one  pure  original  race  grown  from  a single  individual.  From 


r 


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a coli-typhosus-group  organism  they  have  obtained  in  pure  culture 
(l)  a true  Gartner  strain,  (a)  a similar  strain,  but  differing  in 
agglutinating  power,  (3)  a typical  typhosus,  and  (4)  a strain  al- 
most identical  with  B.  coli  mutabile  (Maseini). 

The  papers  of  Burri  and  Duggeli  (13)  and  Burri  and  And- 
re;) ew  (12)  may  be  considered  as  parts  of  Burri' s admirable  paper 
of  1910  (ll).  Burri  (ll)  has  isolated  a race  of  organisms  of  the 
coli-typhosus  group  which  are  unable  to  ferment  saccharose  and 
lactose.  He  calls  this  race  Bacterium  imperfectum.  It  never  ac- 
quired the  power  of  splitting  lactose,  but  on  the  other  hand, 
when  grown  in  media  containing  saccharose,  some  colonies  acquired 
the  power  of  splitting  this  sugar. 

Burri’s  (ll)  observations  were  originally  made  upon 
organisms  grown  in  "shake  cultures"  and  not  on  Endo's  medium.  The 
cultures  were  made  in  the  ordinary  way  and  also  from  single  indivi- 
duals isolated  by  his  Indian  ink  method.  Each  non-saccharose 
splitting  mutating  organism  was  found  to  give  rise  to  only  a very 
low  percentage  of  saccharose-splitting  colonies.  The  latter,  how- 
ever, had  acquired  the  power  permanently,  and  always  retained  this 
character  in  following  generations,  even  after  any  number  of  sub- 
cultures in  many  different  media. 

By  a number  of  ingenious  experiments  Burri  (11 ) attempt- 
ed to  determine  what  percentage  of  the  offspring  of  B.  imperf ectum 
underwent  mutation  into  B.  perfectum.  He  concludes  that  all  of 
the  individuals  of  the  imperf ectum  race  are  able,  under  suitable 


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cultural  and  environmental  conditions,  to  mutate  into  perfectum 
forms. 

In  another  set  of  experiments  he  has  shown  that  the 
change  from  the  imperfectum  to  the  perfectum  type  is  not  a sudden 
change  but  a rather  gradual  one.  Between  the  non-saccharose-spl it- 
ting  imperfectum  and  the  saccharose-splitting  perfectum  intervene 
many  generations  of  individuals  showing  every  transitional  stage. 
This  power  once  acquired  by  a race  is  never  lost. 

Burri  (ll)  supposes  that  the  power  to  ferment  saccharose 
is  latent  in  every  B.  imperfectum  individual,  probably  in  the  form 
of  a zymogen  or  pro-ferment  of  some  sort.  The  enzym  is  produced 
gradually  by  the  constant  action  of  the  sugar  on  successive  genera- 
tions. He  thinks  that  the  newly  acquired  power  of  attacking  sac- 
charose does  not  represent  a regeneration  of  a power  originally 
present  in  the  race  but  temporarily  lost. 

Bernhardt  and  Markoff  (5)  obtained  a col i-typhosus-like 
organism  which  was  capable  of  fermenting  lactose,  but  in  which, 
after  culturing  for  some  time,  and  passage  of  the  organism  through 
mice  and  rabbits,  the  power  of  fermenting  lactose  was  lost.  Thus 
in  this  case  it  would  seem  that  a lactose-fermenting  organism  which 
usually  remains  constant  has  reverted,  or  lost  this  power. 

t 

Barethlein  (3)  found  a very  similar  case  to  that  above 
cited  in  several  races  of  the  coli-typhosus  group:  viz  that  many 
of  the  lactose-splitting  organisms,  when  grown  for  some  time  on 
ordinary  media,  lost  the  power  of  fermenting  lactose.  His  results 


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'W$l 


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seem  to  disagree  with  those  of  most  other  writers.  The  reason 
for  this  may  be  that  he  was  dealing  with  different  races  of  appar- 
ently the  same  organism  which  do  not  all  behave  alike. 

Revis  (30)  in  studying  certain  organisms  of  the  coli- 
typhosus  group  which  produce  both  acid  and  gas  when  grown  on  pep- 
tone broth  containing  lactose,  was  gradually  able  to  acclimatize 
these  organisms  to  grow  in  a medium  containing  0.1  per  cent  mala- 
chite green.  These  organisms  completely  lost  their  power  to  form 
gas  in  the  original  medium  although  they  still  produced  acid.  The 
dye  appears  therefore  to  have  made  a lasting  change  in  their  method 
of  attacking  certain  food  substances. 

Wolf  (45)  carried  out  a.  number  of  extensive  experiments 
to  induce  mutation  in  pigment  production.  He  found  that  the  same 
chemical  substance  often  produced  several  kinds  of  mutations.  Thus 
mercuric  chloride  produced  a dark  red  race  and  a pure  white  race 
in  B.  prodidgiosue . Here  the  mutation  seems  to  have  been  in  oppo- 
site directions;  in  one  case  the  intensifying  of  color,  and  in  the 
other  the  loss  of  color.  Similarly  cadmium-nitrate  produced  a con- 
stant dark  red  mutant  and  a white  reverting  strain  which  did  not 
remain  constant  in  following  generations. 

Eisenberg  (17)  by  cultivating  Bacillus  anthracis  on 
glycerin-agar  obtained  a race  of  organisms  which  did  not  produce 
spores  and  which  remained  constant  in  this  character. 

Barber  (2)  in  studying  Bacillus  coli  found  that  there 


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-8- 


were  certain  long  individuals,  longer  than  the  ordinary  B.  coli. 
constantly  present  in  his  cultures.  By  isolating  and  growing 
these  individuals  he  found  that  the  greater  number  of  them  pro- 
duced colonies  of  individuals  typically  like  B.  coli;  however,  in 
a single  instance  he  obtained  one  which  retained  the  character  and 
gave  only  long  individuals.  It  seems  from  his  results  that  the 
long  individuals  were  of  two  kinds,  though  outwardly  more  or  less 
alike.  The  majority  represented  merely  temporary  modifications 
which  did  not  produce  like  individuals  in  the  following  generations 
while  only  a few  were  permanently  mutated  organisms. 

Revis  (31)  maintains  that  by  growing  B ♦ coli  on  peptone 
broth  to  which  malachite  green  had  been  added,  he  produced  a strain 
which  was  structurally  different,  as  well  as  morphologically  differ 
ent,  from  the  original  culture.  His  experiments  were  not  carried 
on  by  isolation  of  single  individuals,  and  although  the  purity  of 
the  culture  is  guaranteed  his  work  is  open  to  some  criticism  and 
should  be  repeated  using  single  individuals. 

Brierley  (8)  has  divided  the  previous  records  of  muta- 
tions in  fungi  into  two  groups.  "In  the  first  are  those  changes 
in  the  genetic  constitution  which  appear  to  bear  a direct  and 
purposive  relation  to  certain  conditions  which  have  operated  dur- 
ing the  development  of  the  organism  or  that  of  the  preceding  gen- 
eration. The  second  category  of  mutations  include  those  cases  in 
which  the  genetic  change  is  apparently  quite  fortuitous,  and  this 
group  ie  divisible  into  two  sub-groups.  In  the  first  of  these 


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the  change  is  associated  with  certain  conditions  which  interfere 
with  the  normal  metabolic  reactions  of  the  organism.  For  example, 
by  treating  the  organism  with  toxic  substances,  or  by  subjecting 
it  to  extremes  of  temperature  or  dessication,  various  intermediate 
morphological  or  physiological  changes  are  induced,  such  as  alter- 
ations in  spore  dimensions  or  coloration,  or  growth  on  standard 
media.  In  the  second  sub-group  the  changes  occur  spontaneously 
under,  so  far  as  may  be  judged,  perfectly  normal  conditions  of 
development.”  The  latter  sub-group  would  include  the  various 
mutant  forms  of  Alternaria  discussed  in  the  present  paper. 

The  best  known  case  of  purposive  mutation,  perhaps,  is 
that  discussed  by  Massee  (25).  In  this  case  Massee  sowed  spores 
of  Trichothecium  candidum  on  leaves  injected  with  a sugar  solution 
and  from  the  growth  thus  secured  inoculated  other  injected  leaves. 
This  was  done  for  several  generations  of  the  fungus,  after  which 
spores  from  the  last  growth  were  sown  on  normal  leaves  and  infec- 
tion obtained.  "This  means  that  after  12  generations  of  the  fungus 
educated  to  grow  in  Begonia  by  means  of  a chemotrophic  substance 
(a  solution  of  cane  sugar)  the  faculty  of  parasitism  had  been  ac- 
quired for  this  particular  host  plant.  By  similar  means  a para- 

sitic  fungus  can  be  induced  to  become  parasitic  on  a new  host." 

Similar  to  the  findings  of  Massee  (25)  are  those  describ- 
ed by  Salmon  (32)  on  the  adaptive  parasitism  of  Erysiphe  gram inis 
by  means  of  growth  upon  bridging  hosts,  or  the  parallel  results 
of  Marshall  Ward  (43),  Freeman  (20),  Freeman  and  Johnson  (21), 


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-10- 


Johnson  (22) , Pole  Evans  (19)  and  others,  obtained  with  various 
rust  fungi.  None  of  the  above  work,  however,  was  carried  on  with 
single-spore  strains  of  the  organism  in  question  and  is  therefore 
open  to  criticism.  The  work  of  the  latter  writers  has  been  contra- 
vened by  the  work  of  Reed  (29),  Stakman  (37),  Stakman  and  Piemeisel 
(38),  Stakman,  Parker  and  Piemeisel  (39),  and  Stakman,  Piemeisel 
and  Levine  (40).  As  a result  of  their  investigations  the  latter 
state;  "The  results  of  the  experiments  with  P.  gram inis  tritici- 
compact i show  that  barley  which  both  theoretically  and  from  the 
results  obtained  by  previous  investigators  might  be  expected  to 
increase  the  infection  range  does  not  do  so.  Even  susceptible 
varieties  of  wheat  do  not  change  the  parasitic  capabilities  of 
the  rust  so  as  to  enable  it  to  attack  a normally  resistant  variety. 
Furthermore  the  rust  does  not  acquire  additional  virulence  when 

associated  for  a long  time  with  a given  host.  Although  it  is 

possible  that  rusts  may  change  and  new  biologic  forms  may  develop, 
it  seems  more  probable  that  the  change  is  either  a very  gradual 
one,  extending  over  long  periods  of  time,  or  that  they  change  by 
mutation.  No  evidence  of  mutation,  however,  was  obtained  in  the 
present  investigation.  The  difference  may  be  one  of  evolution  as 
compared  with  experimentally  induced  change.  For  practical  pur- 
poses, however,  it  seems  perfectly  safe  to  say  that  no  certain 
and  marked  changes  in  biologic  forms  need  be  expected  as  a result 
of  growing  on  bridging  hosts ; nor  does  it  seem  probable  that 
biologic  forms  are  able  to  gradually  adapt  themselves  to  semi- 


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-11- 


congenial  hosts  by  constant  association  with  those  hosts.  The 
writers  unsuccessfully  tried  to  get  evidence  of  such  adaptation.” 

The  records  of  instances  of  mutation  in  fungi  induced 
by  exposure  to  unfavorable  conditions  are  few  in  number.  Arci- 
chovskij  (l)  obtained  a form  of  Aspergillus  niger  with  yellow- 
brown  spores,  which  rose  in  a pure  culture  of  the  black  form  grow- 
ing in  Haulin' s fluid  to  which  0.0001  per  cent  of  zinc  sulphate 
had  been  added.  This  form  produced  spores  more  quickly  and  was  of 
faster  growth  than  the  original  culture.  He  mentions  that  he  has 
carried  the  form  through  a number  of  generations,  but  does  not 
make  clear,  whether  it  was  grown  on  a medium  containing  zinc,  or 
on  a normal  medium. 

Schiemann  (34)  obtained  various  mutants  of  Aspergillus 
niger  from  a single-spore  culture  by  growth  on  media  containing 
potassium  bichromate,  and  by  exposure  to  extreme  temperatures. 

The  potassium  bichromate  was  added  in  a concentration  of  1 to  2,000 
and  1 to  20,000  parts  to  a medium  prepared  from  cane-sugar,  pep- 
tone, potassium  nitrate,  magnesium  sulphate  and  potassium  di-hydro- 
gen phosphate.  From  the  first  generation  of  a culture  on  a medium 
containing  potassium  bichromate,  1 to  2,000,  a brown  form  was  iso- 
lated and  cultured  on  malt-agar.  This  mutant  remained  constant 
through  32  generations.  From  the  11th  generation  of  a culture 
growing  on  a medium  containing  potassium  bichromate,  1 to  20,000, 
a form  was  isolated  which  varied  from  a dirty  white  to  a bright- 
brown  in  color.  This  mutant  remained  constant  through  23  genera- 


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tione  on  malt-agar.  From  a culture  growing  on  malt-agar  slants 
free  from  any  poisonous  substance,  but  exposed  to  a maximum  temper- 
ature of  44°  - 45°  C. , a deviating  growth  was  produced.  The  colony 
of  this  form  was  of  faster  growth  than  the  original.  It  had  a 
loose  mycelium  with  many  aerial  hyphae  and  very  long  conidiophores; 
(3-4  mm.  as  compared  to  1-2  mm.  of  the  original).  This  mutant  re- 
mained constant  through  17  generations  when  cultured  on  malt-agar 
at  normal  temperature. 

The  brown  mutant  appeared  three  times;  twice  in  the  med- 
ium containing  potassium  bichromate  and  once  in  a culture  which 
had  neither  been  exposed  to  high  temperature  nor  contained  potass- 
ium bichromate.  Thus  it  seems  that  mutation  occurs  both  with  and 
without  these  unfavorable  conditions  for  growth. 

Waterman  (44)  by  cultivating  Penicillium  glaucum,  of  a 
supposedly  pure  race,  in  the  presence  of  p-oxybenzoic  acid,  pro- 
tocatechetic  acid,  salicylic  acid  and  trichloracrylic  acid  was 
able  to  induce  mutation.  The  mutant  thus  produced  was  distinctly 
lighter  in  color,  when  grown  on  malt-gelatin,  in  contrast  to  the 
original  culture.  It  differed  also  from  the  primitive  form  in 
having  fewer  spores,  a decidedly  different  odor,  as  well  as  a 
slowrer  rate  of  growth.  In  continued  cultivation  on  malt-agar  the 
mutant,  which  in  all  cases  seemed  the  same,  remained  constant. 

He  also  found  that  a pure  culture  of  Aspergillus  niger 
when  grown  for  some  timie  in  a 2 per  cent  solution  of  galactose 
gave  three  distinct  forms;  namely,  the  original  black  form,  a 


■ 


v.  raofe*  - I 


T . ’ I e-.T 


. 


. 


■ 


* 


-13- 


brown  form  and  a white  one.  The  brown  and  white  forme  when  isola- 
ted on  malt-agar  gave  fewer  spores  than  the  original,  and  the  white 
form  fewer  than  the  brown  one.  There  was  also  a decrease  in  the 
color  intensity  of  the  spores  in  the  mutants,  the  spores  being 
brown  instead  of  black.  There  was  also  a difference  in  the  metab- 
olism of  the  original  and  the  mutants,  the  latter  showing  a more 
vigorous  combustion  of  p-oxybenzoic  acid  to  carbonic  acid.  In 
this  respect  the  white  form  had  a more  vigorous  combustion  than 
the  brown  one. 

Brierley  (8)  reports  that  he  has  tried  carefully  to 
repeat  the  above  experiments  of  Arcichovskij  (l),  Schiemann  (34) 
and  Waterman  (34),  using  single-spore  cultures  of  Aspergillus 
niger  and  Penicillium  italicum.  He  was  unable  to  do  so  and  states, 
"Although  modifications  in  the  color  of  the  spores,  general  morph- 
ological facies,  and  physiological  reactions  were  observed,  these 
affected  the  whole  growth  acted  upon  to  an  equal  degree;  but  the 
reproductive  bodies  of  the  modified  fungi,  gave  in  every  case  the 
original  result.  These  changes  were  phenotypic  and  not  genotypic 
and  throughout  the  whole  series  of  experiments  not  a single  muta- 
tion occurred. " 

Stevens  (41)  tested  the  effect  of  the  amount  of  the 
medium,  humidity,  temperature  extremes,  and  content  of  the  medium 
upon  cultures  of  Helminthosporium.  He  found  that,  although  there 
were  variations  produced  by  these  environmental  changes,  they  were 
mere  modifications  and  did  not  remain  constant  when  the  fungus  was 


' 


. 


I 

. 


. 


. 


-14- 


returned  to  favorable  environmental  conditions. 

Crabill  (14)  attempted  in  four  ways  to  induce  mutation 
artificially  in  Coniothyrium  pirinum  but  without  success. 

The  literature  dealing  with  the  general  question  of  muta- 
tion in  the  Eumycetes  has  been  fully  discussed  by  Stevens  (41), 
and  it  is  not  deemed  necessary  to  go  into  a detailed  review  of 
these  papers  here.  There  are,  however,  a few  which  he  has  not 
mentioned  and  some  which  have  a more  or  less  direct  bearing  upon 
the  present  problem  which  deserve  some  consideration. 

Crabill  (14)  working  with  single-spore  cultures  of 
Coniothyrium  pirinum  described  two  strains  which  he  designated  as 
plus  and  minus.  These  strains  differed  markedly  in  several  charac- 
ters, particularly  in  size  and  abundance  of  pycnidia,  and  in  color 
of  colony.  He  found  in  his  different  strains  variations  ranging 
from  many,  large,  fully  developed  pycnidia  to  few  small  pycnidia, 
verging  on  complete  sterility.  He  says;  "The  cultural  studies 
show  that  minus  strains  may  rise  from  plus  strains  by  sudden  sport- 
ing or  mutation.  An  objection  might  be  raised  that  these  cultures 
were  impure,  i.e.  mixtures  of  two  strains.  In  anticipation  of 
such  an  idea  it  seems  desirable  to  state  that  frequent  pourings  of 
dilution  cultures  were  used  to  preclude  such  a possibility.  Pro- 
geny were  then  selected  only  from  well-isolated  plants,  microscopic 
examination  of  which  showed  that  each  had  been  derived  from  a 
single  spore. — - Both  strains  have  repeatedly  arisen  from  the  pro- 
geny of  a single  plus  spore.  When  once  purified  the  minus  strain 


-15- 


remains  constant  from  generation  to  generation.  The  variation  is 
apparently  occurring  in  only  one  direction.  The  only  explana- 

tion which  remains  is  that  the  minus  strain  is  a sport  or  mutant 
arising  from  the  plus  strain  at  irregular  and  unprognosticable 
intervals. " 

The  plus  strains  by  sudden  variation  give  rise  to  the 
minus  strains  but  the  minus  strains  were  not  seen  to  give  rise 
to  the  plus  strains.  Contrary  to  the  findings  of  Stevens  (41)  and 
myself  he  states  that  the  variation  apparently  occurs  in  the  spores 
and  not  in  the  mycelium.  He  pictures  sectoring  and  colony  differ- 
ences quite  similar  to  those  shown  by  Stevens  (41)  for  Helmintho- 
sporium  and  those  pictured  in  this  paper  for  Alternaria. 

Burger  (9)  in  studying  Colleto trichum  gloeosporioides 
found  variant  sectors  occurring  in  certain  of  his  cultures.  Upon 
transferring  mycelium  from  these  sectors  he  obtained  a strain  dis- 
tinctly different  from  the  original  in  morphological  cultural 
characters.  Since  the  sectoring  was  of  common  occurrence  in  his 
cultures  he  thought  of  the  possibility  of  mixed  cultures  and  grew 
colonies  from  single  spores.  He  found  that  the  sectoring  contin- 
ued and  states;  "One  might  be  led  to  conclude  from  the  foregoing 
data  that  Colletotriohum  gloeosporioides  is  constantly  giving  off 
new  types  under  natural  conditions,  as  well  as  in  artificial  cul- 
tures. 

Brierley  (8)  has  reported  an  albino  mutant  of  Botry tia 


cinerea. 


This  strain  developed  by  forming  a colorless  eclerotium 


. 

; 


-16- 


in  a culture  which  normally  formed  black  sclerotia.  The  parent 
colony  originated  from  a single  spore  and  had  been  under  cultiva- 
tion for  some  time.  Brierley  states  in  summary:  "The  colorless 
form  arose  spontaneously  without  any  evident  relation  to  external 
conditions  or  stimuli,  in  a single  sclerotium  of  a culture  which 
on  accepted  criteria  was  a pure  line.  The  new  form  is  apparently 
differentiated  from  its  parent  in  respect  to  a single  character 
only,  that  of  color,  and  this  albinism  would  appear  to  be  associa- 
ted with  the  absence  of  cromogen.  The  change  has  occurred  once 
only,  and  has  given  rise  to  a form  unknown  in  nature  and  perfectly 
constant  under  all  conditions.  It  would  seem  possible  to  place 
only  one  interpretation  on  these  facts,  that  of  mutation,  and 
accordingly  when  I was  privileged  to  exhibit  this  form  to  the 
Linnean  Society  of  London  on  April  3,  1919,  I described  it  as  'an 
albino  mutant  of  Botrytis  cinerea.'" 

Blakeslee  (6)  reports  numerous  variations  in  single- 
spore cultures  of  Mucor.  Many  of  these  mutants  tend  to  revert  to 
the  original  form  when  cultured;  however,  he  has  found  two  mutants 
which  are  more  stable  and  maintain  the  varying  characteristics. 

Stevens  (41 ) while  studying  a species  of  Helminthospor- 
ium  causing  a foot-rot  of  wheat  noticed  the  prevalence  of  sectors 
of  different  color  and  growth  occurring  in  his  cultures.  Single- 
spore cultures  of  the  original  culture  were  made  and  carefully 
studied.  He  found  that  saltation  occurred  quite  frequently  in 
this  species.  The  saltants  differed  from  the  original  strain  in 


' 


■ 

. 


■ 


-17- 


a number  of  ways,  particularly  however,  in  color  of  colony,  rate 
of  growth,  number  of  spore  septatione  and  length  of  spores.  The 
case  he  describes  for  Helminthosporium  and  the  pictures  and  plates 
presented  are  quite  similar  to  those  presented  for  this  paper. 

There  exists  among  the  various  writers  on  the  mutation 
question  differences  as  to  the  usage  of  the  term  mutation.  The 
bacteriologists  denote  (Dobell  (16))  " a permanent  change  - how- 
ever small  it  may  be  - which  takes  place  in  a bacterium  and  is 
then  transmitted  to  subsequent  generations.  All  other  changes 
which  are  impermanent  depending  generally  upon  changes  of  the  en- 
vironment - and  hereditarily  fixed,  are  called  modifications." 
Brierley  (7)  defines  mutation  as  "a  genotypic  change  in  a pure 
line".  In  the  present  paper  the  term  mutation  is  used  to  denote 
a sudden  morphological  change  which  is  transmittable  to  subsequent 
generations.  In  this  respect  it  does  not  necessarily  imply  a 
nuclear  change,  and  is  similar  to  the  terms  saltation  as  used  by 
Stevens  (41),  or  mutation  as  used  by  Blakeslee  (6),  Grabill  (14) 
and  Burger  (9). 

From  the  above  summaries  of  the  mutation  question  in 
bacteria  and  fungi  one  may  draw  certain  conclusions.  If  we  consid- 
er that  the  work  of  the  above  writers  has  been  carefully  done,  then 
we  must  conclude  that  mutations  occur  under  cultural  conditions 
and  it  is  reasonable  to  suppose  that  such  phenomena  occur  in  nature. 
Many  of  the  above  papers  are  undoubtedly  open  to  criticism,  espec- 
ially those  dealing  with  bacteria,  because  of  the  loose  methods 


. 


. 


, 


' 


. 


- 


-13- 


employed  or  conclusions  drawn  from  too  scanty  evidence.  However, 
in  such  work  as  that  of  Burri  (11)  and  Barber  (2),  both  of  whom 
worked  with  single-spore  cultures  and  carried  on  their  work  under 
the  best  of  conditions  it  can  hardly  be  denied  that  mutation  has 
taken  place. 

Brierley  (7)  is  not  inclined  to  accept  the  recorded  mu- 
tations for  bacteria  on  the  grounds  that  little  or  nothing  is 
known  of  the  genetic  constitution  of  bacteria,  and  also  since  the 
strains  in  question  were  not  derived  from  pure  lines.  He  looks 
upon  the  variations  recorded  as  being  mere  segregations  of  hetero- 
gynous  strains  and  not  mutations  in  his  sense  of  the  term. 

A possible  method  by  which  these  strains  might  be  of 
mixed  genetic  constitution  has  been  brought  out  by  the  work  of 

LOhnis  and  Smith  (24)  on  the  existence  of  life  cycles  in  bacteria. 

/ 

They  state:  ”The  life  cycle  of  each  species  studied  is  composed 
of  several  sub-cycles  showing  wide  morphological  and  physiological 
differences.  They  are  connected  to  each  other  by  the  symplastic 
stage.  Direct  changes  from  one  sub-cycle  into  another  occur,  but 
they  are  rather  rare  exceptions.”  In  this  respect  it  is  not  in- 
conceivable that  in  the  bacterial  life  cycle  there  may  exist  some 
phase  more  or  less  comparable  with  the  sexual  fusions  of  other 
organisms.  LChnis  and  Smith  (24),  for  example  note  a 83'Tnplastic 
stage  in  which,  ”the  living  matter  previously  inclosed  in  the 
separate  cells  undergoes  a thorough  mixing  either  by  a complete 
disihtegrat ion  of  the  cell  walls,  as  well  as  cell  content,  or  by 


■ 

\ 


-19- 


the  melting  together  of  the  contents  of  many  cells  which  leave 
their  empty  cell  walls  behind  them".  Furthermore  they  state,  " 
another  mode  of  interaction  between  the  plaemic  substance  in 
bacterial  cells  was  obeerved  consisting  in  the  direct  union  of 
two  or  more  individual  cells.  This  conjunction  seems  to  be  of 
no  lees  general  occurrence  than  the  process  first  mentioned". 

Such  conditions  as  these,  if  existing  in  the  bacteria  may  offer 
an  explanation  of  the  apparent  mutations  in  bacteria. 

The  suggestion  has  been  offered  that  the  mutations  and 
saltations  of  Crabill  (14),  Burger  (9),  Stevens  (41)  were  due  to 
segregation  in  a heterozygous  strain.  It  is  true  that  in  all 
these  cases  cultural  work  was  begun  from  a single  asexual  spore 
which  to  all  external  appearances  was  homozygous.  Yet  it  is  true 
for  some,  and  possibly  true  for  all,  that  an  ascigerous  or  sexual 
stage  exists.  It  is  also  conceivable  that,  if  the  spores  from 
which  these  cultures  originated  had  been  derived  in  the  not  far 
distant  past  from  an  ascigerous  stage,  the  possibility  of  their 
being  he tercz3rgous  exists.  Stevens  (4l)  states,  "this  possibility 
is  purely  hypothetical  but  it  appears  to  offer  a possible  explana- 
tion for  the  saltations  in  Helminthosporium".  Dastur  (15)  found 
variation  in  Gloeosporium  piperatum  a common  occurrence  only  in 
strains  recently  derived  from  perithecia.  This  fact  would  tend 
to  support  such  an  hypothesis. 

It  may  be  that  seme  of  my  cultures  of  Alternaria  which 
are  mutating  have  been  recently  through  a sexual  stage,  while 


• 

* 


* 


' 


j 


-20- 


others  which  show  no  signs  of  mutation  have  not.  All  of  my  cul- 
tures have  been  secured  from  asexual  spores  and  none  of  them  have 
shown  signs  of  producing  perithecia.  One  point  against  such  an 
argument  is  the  fact  that  my  culture  No.  1 which  has  been  under 
constant  cultivation  for  over  14  months  and  during  that  time  has 
been  grown  three  times  from  single  spores  still  continues  to  mu- 
tate. 

Brierley  (8)  offers  entirely  different  suggestions  as  to 
the  possible  explanation  of  his  mutation  in  Botrytis  cinerea,  in 
which  sexuality  is  unknown.  He  suggests  the  possibility  of  a nu- 
clear transference  due  to  anastomosis  of  the  mycelia,  or  the  cyto- 
plasmic contamination  by  such  anastomosis.  He  presents  no  evidence 
for  his  suggestions,  and  although  anastomosis  is  common  among 
fungi  it  would  take  detailed  cytological  study  to  prove  a nuclear 
transference  or  fusion. 

III.  Source  of  Materials 

The  strains  or  species  of  Alternaria  used  in  these 
studies  have  been  secured  from  widely  different  and  varied  sources. 
Strain  1 was  obtained  from  germinating  millet  purchased  from  a 
Champaign  seed-house.  Strain  7 was  isolated  from  a ripe  wheat- 
head  from  Granite  City,  Illinois.  Strain  10  was  secured  from  dead 
onion  seed— stalks  from  Crawf ordsville,  Indiana.  Strain  15  was  iso- 
lated from  decaying  applee  obtained  from  a grocery  in  Urbana. 

Other  etraine  which  have  failed  to  show  signs  of  mutation  have 
been  isolated  and  studied  from,  leaf-spots  on  Datura,  Lilium  and 


. 

■ 


■ ' . H 

. 


-21- 


Yucca,  from  fruits  of  okra,  morning  glory,  snow  berry,  mango  and 
pepper,  and  from  dead  blades  of  grass  and  corn. 

IV.  Methods  Employed  for  Isolation  and  Growth 
Cultures  of  Alternaria  from  the  different  sources  were 
usually  obtained  by  planting  infected  tissue  of  the  host  on  agar, 
or  by  scraping  spores  from  the  host  into  a few  drops  of  sterile 
water  which  was  then  added  to  a tube  of  agar  and  poured  into  a 
sterile  Petri-dish.  The  growth  thus  secured  was  examined  and  trans- 
ferred until,  as  far  as  could  be  ascertained,  a pure  culture  had 
been  obtained.  Single-spore  cultures  were  then  made  in  order  to 
be  sure  that  the  cultures  were  pure.  This  was  accomplished  in  the 
following  manner.  Spores  from  the  cultures  were  transferred  to  a 
tube  containing  about  10  cubic  centimeters  of  sterile  water.  It 
was  quite  essential  that  only  a few  spores  be  placed  in  the  water. 

In  order  to  ascertain  the  approximate  number  of  spores  per  oese  in 
the  suspension,  two  oeses  were  transferred  to  a slide  and  examined 
under  the  microscope.  If  the  number  of  spores  thus  obtained  was 
under  12  the  suspension  was  considered  satisfactory.  Two  oeses 
of  the  suspension  were  then  transferred  to  a tube  of  melted  agar 
at  42°  C.  , thoroughly  mixed  by  rolling  between  the  hands,  and 
poured  into  a sterile  Petri-dish.  As  soon  as  the  agar  had  hardened 
the  poured  plates  were  examined  by  inverting  them  under  the  low 
power  of  the  microscope.  In  this  way  single  spores,  well  isolated 
from  other  spores,  were  located  and  their  position  marked  by  draw- 
ing a circle  around  them  with  Indian  ink,  on  the  bottom  of  the 


. 

. 


. 


. 


-22- 


Petri-dish.  Great  care  was  used  in  making  sure  that  only  one  spore 
was  inside  the  circle.  The  agar  within  the  circle  was  carefully 
lifted  out,  transferred  to  another  sterile  agar  plate  and  again 
examined  to  make  sure  that  only  one  spore  had  been  transferred. 

The  single  spore  was  then  allowed  to  germinate  and  produce  a colony. 

Transfers  from  the  colonies  originating  from  single  spores 
have  been  made  to  plates  of  corn-meal  agar  and  grown  at  room  temper- 
ature to  observe  whether  mutation  would  take  place  or  not.  In  all 
the  cultural  work  corn-meal  agar,  made  by  the  following  formulae, 
has  been  used. 

Fifty  grams  of  corn-meal  were  added  to  1,000  cc.  of  dis- 
tilled water  and  cooked  for  one  hour  over  a water  bath  at  60°  C.  , 
then  filtered  through  cloth  and  filter  paper  and  13  grams  of  agar 
shreds  added.  The  above  medium  was  then  cooked  for  one  and  one- 
half  hours  over  a water  bath,  filtered  through  cotton,  tubed  and 
sterilized  in  the  autoclav  for  20  minutes  at  fifteen  pounds  pressure. 

Care  has  been  taken  to  have  the  media  as  nearly  constant 
as  possible  to  avoid  variations  due  to  the  quality  of  the  medium. 

Appearance  of  Mutations 

The  mutations  herein  reported  always  arose  as  sectors 
differing  in  color  from  that  part  of  the  parent  colony  adjacent  to 
them.  Transfers  were  made  from  these  variant  sectors  to  poured 
sterile  agar-plates,  and  a transfer  of  the  normal  colony  placed  in 
the  same  plate  some  distance  from  the  variant  transfer.  The  variant 
transfer  was  marked  as  M and  the  normal  transfer  designated  as  0. 


* 


. 

' 


. 

* 


. 


4 p 


-23- 


The  colonies  produced  from  such  transfers  were  allowed  to  grow 
until  they  filled  the  plate  when  other  transfers  were  made,  and 
so  on  for  a large  number  of  generations.  Ey  this  method  it  was 
possible  to  study  the  mutant  and  the  original  under  approximately 
the  same  conditions.  The  two  colonies  grown  in  this  way  differed 
most  markedly  in  color,  presence  or  absence  of  aerial  mycelium, 
and  in  conidial  production.  In  practically  all  cases  such  mutants 
arising  from  distinct  sectors  have  remained  constant,  though  occa- 
sionally one  is  obtained  which  in  turn  gives  off  sectors  which  are 
apparently  reversions  to  the  original  form.  In  the  contrast  stud- 
ies of  the  mutants  and  the  originals  care  has  been  taken  that  the 
thickness  of  the  medium  be  the  same  all  over  the  place,  since  it 
was  found  that  the  thickness  of  the  medium  plays  an  important  part 
in  the  appearance  of  the  colony. 

Throughout  the  paper  M denotes  a mutant  while  0 denotes 
the  original  culture.  I have  given  the  different  strains  of 
Alternaria  tested  for  mutation  a serial  number.  Thus  0 10,  M2 
refers  to  a mutant,  No.  2,  arising  from  original  culture  No.  10. 

A generation  is  taken  to  mean  the  growth  produced  on  a sterile 
plate  by  a transfer  of  mycelium  from  another  culture. 

In  original  culture  No.  10  mutant  1 appeared  in  the  2nd 
generation.  This  culture  has  been  giving  rise  to  mutants  for  more 
than  14  months.  Some  18  of  these  mutants  have  been  isolated  and 
studied  but  they  appear  to  be  the  same,  or  ver}*-  closely  alike, 
and  are  similar  to  mutant  1.  Mutant  15  appeared  in  the  22nd  gener- 
ation of  the  original  culture  and  in  the  4th  generation  after  re- 


. 


. 


' . • 


-24- 


isolation  from  a single  spore. 

In  culture  No.  7 mutant  1 appeared  in  the  12th  genera- 
tion. This  mutant  has  appeared  but  once  and  has  repeatedly  given 
off  sectors  which  appear  to  be  reversions  to  the  original,  since 
no  significant  differences  are  brought  out  by  contrast  studies. 

In  culture  No.  10  two  distinct  mutants  have  been  iso- 
lated from  among  the  many  variant  sectors  given  off  by  the  original 
culture.  Mutant  1 appeared  in  the  4th  generation  of  the  original 
culture;  mutant  2 in  the  9th  generation.  Other  mutants  have  arisen 
from  this  culture  but  in  contrast  studies  have  not  appeared  to  be 
distinct  from  mutant  1.  Thus  it  might  be  said  that  mutant  1 has 
appeared  several  times.  Mutant  2 has  arisen  but  once  and  has  re- 
mained constant. 

Modifications 

Throughout  these  cultural  studies  certain  modifications 
have  arisen.  The  most  common  modifications  are  in  the  form  of 
small  sectors  of  different  color  at  the  very  edge  of  any  old  colony 
which  is  drying  out.  In  most  cases  transfers  from  these  small 
sectors  give  normal  colonies  like  the  original.  A form  of  modi- 
fication less  common  than  the  above  is  the  appearance  of  light  or 
dark  radiating  streaks  in  original  cultures  Nos.  7 and  10.  Trans- 
fers made  from  these  streaks  produce  normal  colonies.  In  a normal 
colony  which  has  grown  to  the  edge  of  the  plate  white  aerial  mycel- 
ium is  often  produced  on  the  sides  of  the  plate.  This  upon  trans- 
fer does  not  maintain  its  characteristic  white  appearance  but  gives 


' 


-25- 


a normal  colony.  In  cases  where  the  medium  is  not  the  same  thick- 
ness throughout  the  plate  certain  modifications  are  produced  which 
do  not  maintain  themselves  when  transferred.  Modifications  have 
been  induced  by  the  addition  of  certain  chemicals  and  toxic  sub- 
stances to  the  medium.  Transfers  from  such  modifications,  grown 
on  a normal  medium,  produce  normal  colonies. 

V.  Methods  Employed  for  Contrast  Studies 
of  Mutants  and  Originals 

In  the  following  studies  for  the  contrast  in  color  of 
colonies,  zonation  of  colonies,  rate  of  growth,  presence  or  ab- 
sence of  aerial  mycelium  and  abundance  of  conidial  production,  the 
mutants  have  been  grown  side  by  side  with  each  other  and  with  their 
parent,  under  practically  the  same  conditions.  For  the  contrast 
of  length  and  breadth,  conidia  were  measured  under  the  microscope 
by  means  of  an  ocular  micrometer  having  a value  of  3.4  mic.  per 
space.  A mechanical  stage  was  used  to  move  the  slide  on  the  micro- 
scope, All  conidia  were  measured  which  came  between  certain  lines 
on  the  ocular  micrometer.  In  this  way  unconscious  selection  of 
conidia  was  avoided.  The  conidia  were  divided  into  classes  and 
plotted  on  cross  section  paper  according  to  the  number  of  ocular 
spaces  occupied.  In  cases  where  conidia  fell  midway  between  spaces 
note  was  made  of  them  and  they  were  equally  divided  between  the 
two  classes.  The  results  of  these  measurements  and  the  results  of 
counting  the  number  of  cross  and  longitudinal  septations  have  been 
plotted  in  the  form  of  polygons  and  curves  and  compared  biometrie- 


-26- 


ally.  Ifr  contrasting  the  color  of  the  conidia,  mycelia  and  media, 
two  Leitz  microscopes  of  approximately  the  same  magnification  were 
used.  The  fields  of  these  two  microscopes  containing  the  desired 
material  were  brought  together  by  using  and  E.  Leitz,  Wetzlar 
comparison  ocular.  In  this  way  the  different  shades  and  tints 
could  be  quite  accurately  compared. 

In  view  of  the  fact  that  much  variation  is  caused  by  the 
quality  of  the  medium,  (Planchon  (28))  the  media  used  in  all  cul- 
tures has  been  made  as  nearly  constant  as  possible.  To  avoid 
differences  due  to  the  age  of  the  cultures,  colonies  of  the  same 
age  have  been  used  in  all  contrast  studies.  By  this  method  it 
has  been  possible  to  add  some  significance  to  the  differences  in 
color  of  spores  and  mycelium,  which  would  not  be  justifiable  if 
cultures  of  unequal  age  had  been  used. 

Color  of  Colonies  on  Agar 

Culture  No.  1. 

The  original  culture  gives  a very  light  grey  qjOLony  with 
white  aerial  mycelium.  Mutant  1 has  a reddish-brown  colony.  Mu- 
tant 15  gives  a gray  colony,  but  much  darker  in  hue  than  the  orig- 
inal, and  it  has  dark  aerial  mycelium. 

Culture  No.  7. 

The  original  colony  of  this  culture  is  lead  colored. 
Mutant  1 is  light-brown  with  a bluish  tint.  The  mutant  is  much 
darker  than  the  original  but  has  not  nearly  as  much  brown  color 
as  mutant  1 of  culture  No.  10,  or  mutant  1 of  culture  No.  15. 


-27- 


Culture  No.  10. 

The  original  culture  has  a colony  that  is  dark  gray 
approaching  black.  Mutant  1 has  a reddish-brown  colony  much  light- 
er in  color  than  the  original.  Mutant  2 gives  a colony  similar 
in  general  color  to  the  original  but  of  brown  tint.  It  is  of  much 
darker  hue  than  mutant  1 but  does  not  have  the  reddish-brown  cast 
of  mutant  1. 

Culture  No.  15. 

The  original  culture  gives  a bluish-gray  colony;  mutant 
1 a reddish-brown  colony  similar  in  appearance  to  mutant  1 of  cul- 
ture No.  1,  but  lighter  in  hue  than  mutant  1 of  culture  No.  7 and 
mutant  1 of  culture  No.  10. 

Zonation  of  Colonies 

Culture  No.  1. 

There  is  no  zonation  in  the  colonies  of  the  original ; a 
slight  zonation  in  the  colonies  of  mutant  1 and  a very  distinct 
zonation  in  the  colonies  of  mutant  15. 

Culture  No.  7. 

Colonies  of  the  original  show  a slight  zonation  and  light 
streaks  radiating  from  the  center.  Colonies  of  mutant  1 show  a 
distinct  zonation  which  is  more  prominent  than  in  the  original. 

The  radiating  streaks  present  in  the  original  are  absent  in  the 
mutant. 

Culture  No.  10. 

Colonies  of  the  original  culture  show  no  marked  zonation. 


. 


. 


-38- 


Colonies  of  mutant  1 show  a distinct  and  marked  zonation,  while 
those  of  mutant  2 show  a slight  zonation  which  ie  not  so  pronounoed 
as  that  of  mutant  1. 

Culture  No.  15. 

Colonies  of  the  original  culture  show  very  slight  zona- 
tion while  those  of  mutant  1 show  distinct  zonation. 

Rate  of  Growth 

Contrast  studies  of  the  different  mutants  and  their 
originals  have  shown  little  or  no  difference  in  their  rate  of 
growth.  There  appears  to  be  a slight  difference  between  original 
15  and  its  mutant  1 in  that  the  mutant  grows  slightly  faster  than 
the  original.  The  difference  here  is  very  slight  and  not  readily 
apparent. 

Color  of  Mycelium 

Culture  No.  1. 

The  original  culture  has  a hyaline  mycelium.  The  mycel- 
ium of  mutant  1 ie  yellowish-brown.  The  mycelium  of  mutant  15  is 
gray  being  much  darker  than  that  of  the  original. 

Culture  No.  7. 

The  mycelium  of  mutant  1 is  thicker  and  slightly  darker 
than  that  of  the  original  culture. 

Culture  No.  10. 

The  mycelium  of  the  original  is  darker  than  that  of 
either  of  the  mutants.  The  original  culture  has  a dark-gray  mycel- 
ium; mutant  1 a nearly  hyaline  mycelium  with  a yellowish  cast;  mu- 


-29- 


tant  2 a light-gray  mycelium. 

Culture  Mo.  15. 

The  mycelium  of  the  original  culture  is  lead-colored 
while  that  of  mutant  1 is  much  lighter  with  a yellowish  cast. 

Color  of  Medium. 

In  cultures  Nos.  1,  7,  and  15  neither  the  originals  nor 

i A 

the  mutants  color  the  medium  which  they  are  growing.  In  culture 
Mo.  10  the  medium  ie  not  colored  by  the  original  culture  or  mutant 
2,  but  is  colored  slightly  yellow  by  mutant  1. 

Aerial  Mycelium 

Culture  Me.  1. 

There  is  an  abundance  of  aerial  mycel ium  produced  by 
the  colonies  of  the  original  culture.  The  colonies  of  mutant  1 
do  not  produce  aerial  mycelium;  those  of  mutant  15  produce  aerial 
mycelium,  but  not  so  abundantly  as  colonies  of  the  original.  The 
mycelium  of  mutant  15  is  quite  flocculose,  a character  entirely 
absent  in  the  original  culture  and  in  mutant  1. 

Culture  No.  7. 

There  is  an  abundance  of  aerial  mycelium  produced  by  the 
original  culture,  together  with  numerous  white  tufts.  Mutant  1 
produces  considerably  less  aerial  mycelium,  and  the  white  tufts 
are  entirely  lacking. 

Culture  Mo.  10. 

The  original  culture  produces  an  abundance  of  aerial 
mycelium;  this  is  lacking  in  mutant  1 and  is  produced  in  less 
abundance  in  mutant  2 than  in  the  original.  The  mycelium  of  mu- 


. 


. 


-30- 


tant  2 is  quite  flocculose,  a character  lacking  in  the  original 
and  in  mutant  1. 

Culture  No.  15. 

The  original  culture  produces  abundant,  long,  aerial 
mycelium.  Mutant  1 produces  some  aerial  mycelium  which  is  short 
and  with  age  comes  to  lie  flat  on  the  suface,  producing  a felt- 
like weft. 

The  following  mathematical  calculations  represent  the 
mean  (M) , standard  deviation  (<r)  and  coefficient  of  variability 
(C.V.)  as  obtained  by  the  formulae  of  Davenport  and  Fietz  in 
Illinois  Agr.  Exp.  Sta.  Bui.  119,  for  the  graphs  listed  on  plates 
1 to  9;  representing  conidial  measurements  of  length  and  width 
and  the  number  of  cross  and  longitudinal  septa.  On  accepted 
criteria  in  comparison  of  the  means  of  graphs  by  this  method  it 
is  assumed  that  if  the  difference  between  the  means  of  two  graphs 
is  greater  than  6 times  the  probable  error,  the  chance  is  19260 
to  1 that  the  two  strains  are  different.  It  has  not  been  thought 
best  to  consider  differences  of  less  than  -6  times  the  probable 
error  to  be  significant. 

Length  of  Conidia 

Culture  No.  1. 


Original. 

Plate  1. 

Mutant  1. 

Plate  1. 

Mutant  15. 

Plate  1. 

Graph  No 

. 1A. 

Graph  No 

. IB. 

Graph  No. 

1C. 

M.  8.73 

.08 

M.  7.38 

.05 

M.  7.50 

.08 

<r . 1.72 

.05 

&.  1.19 

.04 

1.88 

.06 

C.V.  19.76 

.69 

C.V.  16.17 

. 55 

C.V.  25.13 

.91 

The  difference  between  the  means  of  graphs  1A  and  IB 


is  17  times  the  probable  error.  The  difference  between  the  means 


I 


. . 


-31- 


of  graphs  1A  and  1C  is  15  times  the  probable  error;  that  of 
graphs  IB  and  1C  only  1.5  times  the  probable  error.  The  graphs 
show  that  there  is  a wide  difference  in  conidial  length  between 
the  original  and  the  mutants,  but  that  there  is  only  a slight 
difference  in  the  conidial  lengths  of  the  mutants. 


Culture  No 

. 7. 

Original. 

Plate  1. 

Mutant  1. 

Plate 

Graph  No. 

5A. 

Graph  No. 

5B. 

M.  6.77 

.07 

M.  7.17 

.08 

C;  1.48 

.05 

fr.  1.84 

.06 

C.V.  21.91 

.77 

C.V.  25.75 

.92 

The  difference  in  the  means  here  is  only  5 times  the 
probable  error  which  shows  no  very  significant  difference  in  the 
conidial  lengths  of  the  original  and  the  mutant. 

Culture  No.  10. 

Original.  Plate  1.  Mutant  1.  Plate  1.  Mutant  2.  Plate  1. 
Graph  No.  9A.  Graph  No.  9B.  Graph  No.  9C. 


M. 

8.84 

.13 

M. 

8.03 

.11 

M. 

10.07 

.12 

CT» 

2.90 

.09 

c T- 

2.44 

.08 

cr 

2.60 

.08 

C.V. 

32.80 

1.21 

C.V. 

30.20 

1.09 

C.V. 

25.34 

.92 

By  comparison  of  the  means  of  graphs  9A  and  9B  we  find 
the  difference  to  be  6 times  the  probable  error;  of  graphs  9B  and 
9C,  15  times  the  probable  error.  The  graphs  thus  show  that  there 
is  a wide  difference  between  the  original  and  the  mutants,  and  be- 
tween the  mutants  themselves,  in  respect  to  conidial  length. 

Culture  No.  15. 


Original. 

Plate  1. 

Mutant  1. 

Plate 

Graph  No.  13A. 

Graph  No.  ' 

13B. 

M.  6.10 

.05 

M.  7.44 

.07 

cr.  1.16 

.03 

<T.  1.48 

.05 

G.v.  19.10 

.66 

C.V.  19.96 

. 62 

-32- 


The  difference  between  the  means  of  graphs  13A  and  13B 
is  19  times  the  probable  error  showing  a wide  difference  between 
the  length  of  the  conidia  of  the  original  and  the  mutant. 

Width  of  Conidia 
Culture  Ho.  1. 

Original.  Plate  2.  Mutant  1.  Plate  2.  Mutant  15.  Plate  2. 
Graph  No.  2A.  Graph  No.  2B.  Graph  No.  2C. 


M.  3.32 

.03 

M.  3.23 

.02 

M.  3.20 

.02 

c r.  . 71 

.02 

O'  . 54 

.01 

O'  . 50 

.01 

C.V.  21.46 

. 75 

C.V.  16.87 

.58 

C.V.  15.93 

.55 

The 

difference 

between  the 

means 

of  graphs  2A  and 

2B 

3 times  the  probable  error;  of  graphs  2A  and  2C,  4 times  the  prob- 
able error;  of  graphs  2B  and  2C  no  difference.  It  is  concluded 
from  the  above  evidence  that  there  is  no  essential  difference  be- 
tween the  original  and  the  mutants,  or  between  the  mutants  them- 
selves, in  regard  to  conidial  width. 

Culture  No.  7. 


Original . 

Plate  5. 

Mutant  1. 

Plate 

Graph  No. 

6A. 

Graph  No. 

6B. 

M.  2.95 

.07 

M.  3.03 

.03 

C r.  . 49 

.01 

Or  . 70 

.02 

C.V.  16.81 

.58 

C.V.  23.31 

. 32 

The  difference  between  the  means  of  graphs  6A  and  6B  is 
only  3 times  the  probable  error  and  is  therefore  not  significant. 
Culture  No.  10. 


Original. 

Plate  6. 

Mutant  1.  Plate  6, 

Mutant  2. 

Plate 

Graph  No.  ! 

10A . 

Graph  No.  10B. 

Graph  No.  : 

IOC. 

M.  3.12 

.03 

M.  3.26  .03 

M.  2.89 

.02 

<T  . 66 

.01 

tr.  .75  .02 

cT  .55 

.01 

C.V.  20.56 

.72 

C.V.  23.19  .82 

C.V.  19.20 

.67 

-33 


The  difference  between  the  means  of  graphs  10A  and  10B 
is  equal  to  3 times  the  probable  error;  that  of  graphs  10A  and 
IOC,  9.5  times  the  probable  error,  and  that  of  graphs  10B  and  IOC, 
12  times  the  probable  error.  This  shows  that  there  is  only  a 
slight  difference  between  the  original  and  mutant  1 in  the  width 
of  conidia,  but  a distinct  difference  between  the  original  and 
mutant  2,  and  between  mutants  1 and  2 in  this  respect. 

Culture  No.  15. 

Original.  Plate  9.  Mutant  1.  Plate  9. 

Graph  No.  14A.  Graph  No.  14B. 


M. 

3.01 

.03 

M. 

3.17 

.02 

<T. 

. 63 

.02 

CT 

. 58 

.01 

c.v. 

21.10 

. 74 

C.V. 

18.43 

.64 

The  difference  between  the  means  of  graphs  14A  and  14B 
is  only  5 times  the  probable  error.  Although  this  is  quite  a 
large  difference  it  is  not  considered  sufficient  to  be  significant. 
Cross  Septa 
Culture  No.  1. 


Original. 

Plate  3. 

Mutant  1. 

Plate  3. 

Mutant  15. 

Plate  3. 

Graph  No. 

3A . 

Graph  No. 

3B. 

Graph  No. 

3C. 

M.  3.24 

.03 

M.  2.88 

.03 

M.  3.08 

.04 

c3\  .74 

.02 

Oh  . 75 

.02 

<T  .87 

.02 

C.V.  22.98 

.81 

C.V.  26.25 

.94 

C.V.  28.55 

1.03 

The  difference  between  the  means  of  graphs  3A  and  3B  is 
12  times  the  probable  error;  of  graphs  3A  and  3C,  5 times  the  prob- 
able error,  and  between  graphs  3B  and  3C,  6 times  the  probable 
error.  This  shows  that  there  is  a distinct  difference  between 
the  original  and  mutant  1,  and  between  mutant  1 and  mutant  15, 


-34- 


but  that  there  is  no  distinct  difference  between  the  original 
and  mutant  15  in  regard  to  the  number  of  cross  septa. 

Culture  No.  7. 


Original. 

Plate  5. 

Mutant  1. 

Plate 

Graph  No. 

7A. 

Graph  No.  1 

7B. 

M.  2.76 

.03 

M.  2.63 

.04 

c T,  . 71 

.02 

<T‘  . 86 

.02 

C.V.  25.95 

.93 

C.V.  32.68 

1.21 

The  difference  between  the  means  of  the  above  graphs  is 
only  3 times  the  probable  error  and  therefore  does  not  show  a dis- 
tinct difference  between  the  original  and  the  mutant  in  number  of 
cross  septa. 

Culture  No.  10. 


Original . 
Graph  No. 

Plate  7. 
11A. 

Mutant  1. 
Graph  11B. 

Plate  7. 

Mutant  2. 
Graph  11  C. 

Plate  7. 

M.  3.39 

.06 

M.  3.20 

.05 

M.  3.86 

.05 

O' . 1.32 

.04 

cr.  1.07 

.03 

<3h  1.18 

.03 

C.V.  38.90 

1.52 

C.V.  33.56 

1.27 

C.V.  30.57 

1.12 

The  difference  between  the  means  of  graphs  11A  and  113 
is  3 times  the  probable  error;  of  graphs  11A  and  11C,  8 times  the 
probable  error,  and  of  graphs  11B  and  11C,  11  times  the  probable 
error.  TLi3  shows  that  there  is  only  a slight  difference  between 
the  number  of  cross  septa  in  the  original  and  mutant  1;  a decided 
difference  between  the  number  in  the  original  and  mutant  2,  and 
between  the  number  in  mutant  1 and  mutant  2. 

Culture  No.  15. 


Original. 

Plate  9. 

Mutant  1. 

Plate 

Graph  No. 

15A. 

Graph  No. 

15B. 

M.  2.65 

. 03 

M.  3.06 

.03 

C7\  . 81 

.02 

<T.  .64 

.02 

C.V.  30.85 

1.06 

C.V.  21.08 

. 74 

-35- 


The  difference  between  the  means  of  graphs  15A  and  15B  is  13.5 
times  the  probable  error  showing  that  there  is  a wide  and  distinct 
difference  in  the  number  of  cross  septa  in  the  original  and  the 
mutant. 


Longitudinal  Septa 
Culture  No.  1. 


Original.  Plate  4. 

Mutant  1. 

Plate  4. 

Mutant  15. 

Plate  4 . 

Graph  No.  4A. 

. Graph  No.  - 

4B. 

Graph  No.  4C. 

M.  1.68  .05 

M.  1.34 

.03 

M.  1.66 

.05 

& 1.06  .03 

cT.  . 81 

.02 

cr . 1.05 

. 03 

C.V.  53.48  2.87 

C.V.  60.82 

2.  70 

C.V.  63.35 

2.86 

The  difference  between  the  graphs  4A  and  4B  is  7 times 
the  probable  error;  of  graphs  4A  and  4C  it  is  within  the  error;  of 
graphs  4B  and  4C  it  is  6 times  the  probable  error.  This  comparison 
shows  that  there  is  a difference  between  the  original  and  mutant  1 
in  the  number  of  longitudinal  septa;  no  difference  in  this  respect 
between  the  original  and  mutant  15,  and  a wide  difference  between 
mutant  1 and  mutant  15. 

Culture  No.  7. 

Original.  Plate  5.  Mutant  1.  Plate  5. 

Graph  No.  8A.  Graph  No.  8B. 


M. 

0. 

62 

.03 

M. 

0.91 

.04 

&. 

0. 

76 

.02 

<T. 

0.85 

.02 

C.V. 

12. 

23 

.41 

C.V. 

9.40 

.31 

The  difference  between  the  means  of  graphs  8A  and  8B  is 
7 times  the  probable  error  which  shows  a difference  between  the 
original  and  the  mutant  in  respect  to  the  number  of  longitudinal 
septa  in  the  conidia. 


-36- 


Culture  Wo.  10. 


Original . 

Plate  8. 

Mutant  1. 

Plate  8. 

Mutant  2. 

Plate 

Graph  Wo. 

12A. 

Graph  Wo. 

12B. 

Graph  Wo. 

12C. 

M.  0.77 

.04 

M.  1.46 

.05 

M.  0.87 

.04 

C r.  0.97 

.03 

1.18 

.03 

<^.  0.91 

. 03 

C.V.  12.50 

.43 

C.V.  8.07 

.40 

C.V.  10.55 

.35 

The 

difference' 

between  the  means  of 

the  graphs 

12A  and 

12B  is  17  times  the  probable  error; 

of  graphs 

12A  and  12C 

, 2.  5 

times  the  probable  error,  and  of  graphs  12B  and  12C,  15  times  the 
probable  error.  This  shows  that  there  is  a distinct  difference 
between  the  original  and  mutant  1 in  respect  to  the  number  of 
longitudinal  septa;  no  distinct  difference  between  the  original 
and  mutant  2,  and  a wide  difference  between  mutant  1 and  mutant 
2 in  this  respect. 

Culture  Wo.  15. 

Original.  Plate  9.  Mutant  1.  Plate  9. 

Graph  Wo.  16A.  Graph  Wo.  16C. 

M.  1.08  .04  M.  1.34  .05 

ch  . 90  03  &.  .95  . 03 

C.V.  84.29  4.42  C.V.  71.35  3.41 

The  difference  between  the  means  of  graphs  16A  and  16C 
is  5 times  the  probable  error  which  is  not  considered  large  enoigh 
to  show  that  there  is  any  distinct  difference  between  the  original 
and  the  mutant  in  respect  to  the  number  of  longitudinal  septa  in 
the  conidia. 

Color  of  Conidia 

Culture  Wo.  1. 

The  conidia  of  the  original  culture  are  quite  dark  with 


-37- 


a yellowish  tint;  those  of  mutant  1 are  lighter  and  more  yellow; 
the  conidia  of  mutant  15  are  darker  than  those  of  mutant  1,  but 
not  as  dark  as  those  of  the  original  culture,  and  are  without  the 
yellowish  tint. 

Culture  No.  7. 

The  original  and  mutant  1 have  dark-brown  conidia,  so 
similar  in  color  that  no  difference  is  discernible. 

Culture  No.  10. 

Conidia  of  the  original  culture  are  light-brown,  showing 
dark-brown  in  mass.  Mutant  1 has  light  yellowish  conidia,  much 
lighter  than  those  of  the  original,  but  appearing  similar  in  mass. 
The  conidia  of  mutant  3 are  darker  than  those  of  the  original  and 
of  mutant  1,  and  appear  nearly  black  in  mass. 

Culture  No.  15. 

The  conidia  of  the  original  culture  are  a light  yellow- 
ish-gray, while  those  of  the  mutant  are  darker  with  a brownish  cast. 

Conidial  Production 

Culture  Nol  1. 

In  mutant  1 of  this  culture  numerous  conidia  are  pro- 
duced; slightly  fewer  in  mutant  15,  and  a smaller  number  in  the 
original  than  in  either  of  the  mutants. 

Culture  No.  7. 

The  original  strain  and  mutant  1 both  produce  conidia 
abundantly  in  culture,  but  mutant  1 produces  a great  many  more 
than  the  original,  since  it  has  more  ccnidiophoree  and  more  conidia 


on  each. 


-38- 


Culture  No.  10. 

Mutants  1 and  2 produce  conidia  in  greater  abundance 
than  the  original  strain.  Mutant  1 produces  more  conidia  than 
mutant  2. 

Culture  No.  15. 

The  original  culture  produces  very  few  conidia  and  as  a 
rule  they  are  formed  either  singly  or  in  twos  or  threes  on  a conid- 
iophore.  Mutant  1 produces  conidia  abundantly  in  long  chains. 

Permanency  of  Mutants 

Mutants  which  have  been  isolated  from  distinct  sectors 
of  the  parent  colony  have  usually  remained  constant  in  character. 
All  of  the  mutants  listed  here  have  been  carried  through  from 
twenty  to  fifty  generations  and  have  remained  constant.  A pecu- 
liarity exhibited  by  mutant  15  of  culture  No.  1 is  that  it  seems 
frequently  to  undergo  mutation  and  produce  sectors  which  when  iso- 
lated and  studied  are  indistinguishable  from  mutant  1 of  the  same 
culture.  The  mutants  from  mutant  15  remain  constant  and  show  no 
further  signs  of  mutation  or  reversion. 

Frequency  of  Mutation 

From  these  studies  it  has  not  been  possible  to  form  any 
definite  opinion  as  to  the  frequency  of  mutation  in  Alternaria. 

In  culture  No.  1 mutation  is  of  very  frequent  occurrence,  some- 
times one  or  more  mutants  appear  in  nearly  every  generation,  again 
several  generations  may  occur  before  another  mutant  is  found. 

This  culture  has  been  under  studv  for  14  months  and  has  been  gin- 


. | 


. 


. 


-39- 


ing  rise  to  mutants  rather  constantly  during  that  time.  The  major- 
ity of  these  mutants  seem  to  be  similar  to  each  other,  or  so  near- 
ly similar  that  my  contrast  studies  have  failed  to  note  differences. 
In  culture  No.  7 mutation  seems  to  be  rather  infrequent,  for  the 
culture  has  been  grown  through  ten  generations  since  the  appearance 
of  mutant  1 without  the  appearance  of  a second  mutant.  Mutation 
is  also  of  infrequent  occurrence  in  culture  No.  15,  it  having  been 
under  supervision  for  more  than  30  generations  with  but  a single 
mutation.  In  culture  No.  10  mutation  is  of  more  frequent  occur- 
rence than  in  either  culture  No.  7 or  No.  15  but  is  not  of  as  fre- 
quent occurrence  as  in  culture  No.  1.  The  mutants  of  this  culture, 
with  the  exception  of  mutant  2,  have  all  been  so  similar  to  mutant 
1 that  no  distinction  has  been  possible. 

VI.  Conclusions 

The  foregoing  contrast  studies  have  shown  that  cultures 
isolated  from  variant  sectors  of  strains  of  Alternaria  grown  from 
single  spores  differ  from  the  original  strain  in  a number  of  morph- 
ological characters.  These  differences  are  most  striking  in  color 
of  colonies  as  grown  on  agar-plates;  in  conidial  length;  presence 
of  aerial  mycelium;  zonation  of  colonies;  conidial  production,  and 
number  of  longitudinal  septa  in  the  conidia.  Differences  which 
are  less  common  are  those  of  color  of  the  mycelium,  color  of  the 
conidia,  width  of  conidia  and  number  of  cross  septations  in  the 
conidia.  These  mutants  retain  such  characteristic  differences 
through  many  generations  without  reverting  to  the  original  form. 
Mutation  occurs,  as  a rule,  in  only  one  direction.  Mutants  tend 


. 

....  . 

. 


. 

. 

. 


. 


-40- 


to  form  a darker  colony,  to  have  a more  prominent  zonation,  to 
have  less  aerial  mycelium  and  to  produce  more  conidi  a than  the 
original  strain.  It  is  noticable  that  where  there  is  a decrease 
in  color  of  the  mycelium  there  is  an  increase  in  color  of  the 
conidia. 

Mutation  takes  place  in  the  cells  of  the  mycelium  rather 
than  in  the  conidia  and  occurs  in  some  strains  and  not  in  others. 
No  attempt  is  made  to  explain  the  cause  of  mutation  or  to  account 
for  its  occurrence  in  some  strains  and  not  in  others.  Since  mu- 
tation occurs  very  frequently  in  Alternaria  growing  in  culture  it 
may  be  assumed  that  it  also  occurs  in  nature,  though  no  means  of 
proof  are  yet  known. 


-41- 


VII.  Literature  Cited 

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5.  Bernhardt,  G.  and  Markoff,  W.  N.  Ueber  raodif ikiti onen 

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6.  Blakeslee,  A.  F.  Mutations  in  Mucors.  Jour.  Heredity 

11:278.  1920. 

7.  Brierley,  W.  Bv  Some  concepts  in  mycology  — an 

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8.  On  a form  of  Botrytis  cinerea  with 

colorless  sclerotia.  Phil.  Trans.  Roy.  Soc. 
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9.  Burger,  0.  F.  Variations  in  Colletotrichum  gloeo- 

sporioides.  Jour.  Agr.  Res.  20:723.  1921. 

10.  Burk,  A.  Mutation  bei  einem  koligruppe  verwandten 

bakterium.  Arch.  f.  Hyg.  65:335.  1908. 

11.  Burri,  R.  Ueber  scheinbar  plfttzliche  neuerwerbung 

eines  bestimraten  garungsvermOgens  durch  bakterien 
der  Coligruppe.  Centr.  f.  Bakt.  II,  28:321.  1910. 

12.  and  Andrejew,  P.  Vergleichende  untersuchung 

einiger  Coli  und  Paratyphussat&mme.  Centr.  f. 

Bakt.  I,  56:217.  1910. 

13.  and  Dtlggeli.  Beitrag  zur  systematik  der 

Coli-aercgenesgruppe , u.  s.  w.  Centr.  f.  Bakt. 

I,  49:145.  1909. 

14.  Crabill,  C.  H.  Dimorphism  in  Coniothyrium  pirinum 

Sheldon.  Am.  Jour.  Bot.  3:449-467.  1915. 


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15. 

Dastur,  J.  F.  Glomerslla  cingulata  (Stoneman) 

Spaulding  and  v.  Sch,  and  its  conidial  forms, 
Gloeosporium  piperatum  E.  and  E.  and  Colletotri- 
cum  nigrum  E.  and  Hals,  on  Chillies  and  Carica 
papaya.  Ann.  Appl.  Biol.  6:345-268.  1930. 

16. 

Dobell,  Clifford.  Some  recent  work  on  mutation  in 
micro-organisms.  II.  Mutations  in  bacteria. 
Jour,  of  Genetics  2:325-350.  1913. 

17. 

Eisenberg,  P.  Untersuchungen  liber  die  varibilitflt 

der  Bakterien.  I.  Uber  sporogene  and  asporogene 
Rassen  der  Mil zbrandbacillus.  Centr.  f.  Bakt. 
I,  63:305.  1913. 

CO 

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Elliott,  J.  A.  Taxonomic  characters  of  the  genera 
Alternaria  and  Macrosporium.  Am.  Jour.  Bot.  4: 
439-476.  1917. 

19. 

Evans,  I.  B.  Pole.  South  African  cereal  rusts  with 
observations  on  the  problem  of  breeding  rust- 
resistant  wheats.  Jour.  Agr.  Sci.  4:95-104.  1911. 

30. 

Freeman,  E.  M.  Experiments  on  the  brown  rust  of 
Bromes  (Puccinia  dispersa).  Ann.  Bot.  16:487- 
494.  1902. 

31. 

Freeman,  E.  M.  and  Johnson,  E.  C.  The  rusts  of 
grains  in  the  United  States.  U.  S.  Dept.  Agr. 
Bui.  Bur.  PI.  Industry  216.  1911. 

33. 

Johnson,  E.  C.  Timothy  rust  in  the  United  States, 
U.  S.  Dept.  Agr.,  Bur.  PI.  Industry  Bui.  224. 
1911. 

33. 

Kowalenko,  A.  Studien  tlber  sogenannte  Mutations- 
ercheinungen  bei  Bakterien  unter  besonderes 
Berucksight igung  der  Einzellenkul tur . Zeit.  f. 
Hyg.  66:377.  1910. 

34. 

Ltthnis,  F.  and  Smith,  N.  R.  Life  cycles  of  the 
bacteria.  Jour.  Agr.  Res.  6:675.  1916. 

35. 

Massee,  G.  On  the  origin  of  parasitism  in  fungi. 
Phil.  Trans.  Roy.  Soc.  B,  197:12.  1905. 

36. 

Massini,  R.  Ueber  einen  Biologischer  Beziehung 

Interessanten  Kolistamm  (Bacterium  coli  mutabile). 
Arch.  f.  Hyg.  61:250.  1907. 

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27.  Mtlller , R.  See  Benecke,  citation  4. 

28.  Planchon,  Louis.  Influence  de  divers  milieux 

chimiques  sur  quelques  champignons  du  groupe 
des  Dematiees.  Ann.  des  Sci . Nat.  Bot.  161: 

Ser.  8,  nos.l,  2,  3,  4.  1900. 

29.  Reed,  G.  M.  Physiological  specialization  of  para- 

sitic fungi.  Mem.  Brooklyn  Bot.  Gard.  1:348- 
469.  1905. 

30.  Revis,  C.  Note  on  the  artificial  production  of  a 

permanently  atypical  B.  coli.  Centr.  f.  Bakt. 

II,  31:1.  1911. 

31.  The  production  of  variation  in  the  physiol- 

ogical activity  of  Bacillus  coli  by  use  of  mala- 
chite green.  Proc.  Roy.  Soc.  B,  85:192.  1912. 

32.  Salmon,  E.  S.  Cultural  studies  with  biological  forms 

of  the  Erysiphaceae.  Phil.  Trans.  Roy.  Soc.  B, 
197:107.  1905. 

33.  Sauerbeck,  E.  Ueber  das  Bacterium  coli  mutabile 

(Massini)  und  coli  Varietaten  tlberhaupt.  Centr. 
f.  Bakt.  I,  50:572.  1909. 

34.  Schiemann,  E.  Mutationen  bei  Aspergillus  niger  van- 

Tieghem.  Zeit.  Indukt.  Abstamm.  u.  Vererbungs- 
lehre.  8:35.  1912. 

35.  Schrtteter  and  Gtltjahr.  Vergleichende  Studien  der 

Typhus-coli-Dysenteriebakterien.  Centr.  f.  Bakt. 
I,  58:577.  1911. 

36.  Sobernheim,  G.  and  Seligmann.  Weitere  Beitrage  zur 

Biologie  der  Enter itisbakterien.  Centr.  f.  Bakt. 
I,  50:134.  1911. 

37.  Stakman,  E.  C.  A study  in  cereal  rusts:  physiological 

races.  Minn.  Agr.  Exp.  Sta.  Bui.  138.  1914. 

38.  Stakman,  E.  C.  and  Pieraeisel,  F.  J.  Biologic  forms 

of  Puccinia  graminis  on  cereals  and  grasses. 

Jour.  Agr.  Res.  10:429-495.  1917. 

39.  , Parker,  J.  H.  and  Piemeisel , F.  J. 

Can  biologic  forms  of  stem  rust  on  wheat  change 
rapidly  enough  to  interfere  with  breeding  for 
rust-resistance.  Jour.  Agr.  Res.  14:111-124. 

1918. 


-44- 


40.  Stakman,  E.  C. , Piemeisel,  F.  J.  and  Levine,  M.  N. 

Plasticity  of  biologic  forms  of  Puccinia  graminis. 
Jour.  Agr.  Res.  15:221-250.  1918. 

41.  Stevens,  F.  L.  The  Helminthosporium  foot-rot  of 

wheat,  with  observations  on  the  morphology  of 
Helminthosporium  and  on  the  occurence  of  salta- 
tion in  the  genus.  111.  Nat.  Hist.  Survey  Bui. 

42.  Twort,  F.  W.  The  fermentation  of  gluco sides  by 

bacteria  of  the  typhoid  coli  group  and  the  ac- 
quisition of  new  fermenting  powers  by  Bacillus 
dysenteriae  and  other  micro-organisms.  Proc. 

Roy.  Soc.  B,  79:329.  1907. 

43.  Ward,  H.  Marshall.  Further  observations  on  the 

brown  rust  of  the  Bromes,  Puccinia  dispersa 
(Erikss. ) and  its  adaptive  parasitism.  Ann. 

My col . 1:132-151.  1903. 

44.  Watermann,  H.  J.  Mutation  in  Penicillium  glaucum 

and  Aspergillus  niger  under  the  action  of  known 
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45.  Wolf,  0.  F.  ?!ber  modif ikat ionen  und  exper imen tell e 

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-45- 


VIII.  Explantation  of  Plates 


Plate  I.  Frequency  curves  of  conidial  lengths. 


Graph  1A. 
Graph  IB. 
Graph  1C. 
Graph  5A. 
Graph  5B. 
Graph  9A. 
Graph  9B. 
Graph  9C. 
Graph  13A. 
Graph  13B. 


Original  of  culture  No.  1. 
Mutant  1 of  culture  No.  1. 
Mutant  15  of  culture  No.  1. 
Original  of  culture  No.  7. 
Mutant  1 of  culture  No.  7. 
Original  of  culture  No.  10. 
Mutant  1 of  culture  No.  10. 
Mutant  S of  culture  No.  10. 
Original  of  culture  No.  15. 
Mutant  1 of  culture  No.  15. 


Plate  II.  Frequency  polygons  of  conidial  widths 
of  original  and  mutants  of  culture  No.  1. 


Graph  2A.  Original. 

Graph  2B.  Mutant  1.  Graph  20.  Mutant  15. 

Plate  III.  Frequency  polygons  of  number  of  cross 
septa  in  conidia  of  the  original  and  mutants 
of  culture  No.  1. 

Graph  3A . Original. 

Graph  3B.  Mutant  1. 

Graph  30.  Mutant  15. 

Plate  IV.  Frequency  polygons  of  number  of  longi- 
tudinal septa  in  conidia  of  the  original  and 
mutants  of  culture  No.  1. 

Graph  4A.  Original. 

Graph  4B.  Mutant  1. 

Graph  40.  Mutant  15. 

Plate  V.  Frequency  polygons  of  width  of  conidia 
and  number  of  cross  and  longitudinal  septa  in 
conidia  of  culture  No.  7. 


Graph  6A. 
Graph  6B. 
Graph  7A, 
Graph  7B. 
Graph  8A. 
Graph  8B. 


Conidial  width  of  original. 

Conidial  width  of  mutant  1. 

Conidial  cross  septa  of  original. 

Conidial  cross  septa  of  mutant  1. 
Longitudinal  septa  of  conidia  in  original 
Longitudinal  septa  of  conidia  in  mutant  1 


. 


. 


. 


. 

. 


• 

4 

* 


' 


-46- 


Plate  VI.  Frequency  polygons  of  conidial  width  of 
mutants  and  original  of  culture  No.  10. 

Graph  10A.  Original. 

Graph  10E.  Mutant  1. 

Graph  IOC.  Mutant  2. 

Plate  VII.  Frequency  polygons  of  number  of  cross 
septa  in  original  and  mutants  of  culture  No.  10. 

Graph  11A.  Original. 

Graph  11B.  Mutant  1. 

Graph  11 C.  Mutant  2. 

Plate  VIII.  Frequency  polygons  of  number  of  longi- 
tudinal septa  in  conidia  of  original  and  mutants 
of  culture  No.  10. 


Graph  12A.  Original. 

Graph  12B.  Mutant  1. 

Graph  12C.  Mutant  2. 

Plate  IX.  Frequency  polygons  of  width  of  conidia 
and  number  of  cross  and  longitudinal  septa  in  the 
conidia  of  the  original  and  mutant  of  culture  No. 15. 


Graph  14A. 
Graph  14E. 
Graph  15A. 
Graph  15E. 
Graph  16A. 

Graph  16E. 


Conidial  width  of  original. 
Conidial  width  of  mutant  1. 

Gross  septa  of  original. 

Cross  septa  of  mutant  1. 
Longitudinal  septa  of  conidia  in 
original . 

Longitudinal  septa  of  conidia  in 
mutant  1. 


Plate  X.  Colonies  of  original  and  mutant  1 of  culture 
10  growing  together  in  the  sarnie  plate;  showing  the 
difference  in  color  of  colony  and  amount  of  aerial 
mycel ium. 


Plate  XI.  Colonies  of  original  and  mutant  1 cf  cult- 
ure 1;  showing  difference  in  color  of  colonies  and 
a variant  sector  in  the  original. 


plate  II. 


PLATE  III . 


PLATE  IV. 


CROSS  SEPTA"' 


PLATE  V. 


PLATE  VI. 


HICK 


PLATE  VII. 


PLATE  VIII. 


PLATE  X. 


PLATE  XI 


